Phenotypic and transcriptomic changes in zebrafish (Danio rerio) embryos/larvae following cypermethrin exposure.

Cypermethrin is one of the widely used type-II pyrethroid and the indiscriminate use of this pesticide leads to life threatening effects and in particular showed developmental effects in sensitive populations such as children and pregnant woman. However, the molecular mechanisms underlying cypermethrin-induced development toxicity is not well defined. To address this gap, the present study was designed to investigate the phenotypic and transcriptomic (next generation RNA-Seq method) impact of cypermethrin in zebrafish embryos as a model system. Zebrafish embryos at two time points, 24 h postfertilization (hpf) and 48 hpf were exposed to cypermethrin at a concentration of 10 µg/L. Respective control groups were maintained. Cypermethrin induced both phenotypic and transcriptomic changes in zebrafish embryos at 48 hpf. The phenotypic anomalies such as delayed hatching rate, increased heartbeat rate and deformed axial spinal curvature in cypermethrin exposed zebrafish embryos at 48 hpf as compared to its respective controls. Transcriptomic analysis indicated that cypermethrin exposure altered genes associated with visual/eye development and gene functional profiling also revealed that cypermethrin stress over a period of 48 h disrupts phototransduction pathway in zebrafish embryos. Interestingly, cypermethrin exposure resulted in up regulation of only one gene, tnnt3b, fast muscle troponin isoform 3T in 24 hpf embryos as compared to its respective controls. The present model system, cypermethrin exposed zebrafish embryos elaborates the toxic consequences of cypermethrin exposure during developmental stages, especially in fishes. The present findings paves a way to understand the visual impairment in sensitive populations such as children exposed to cypermethrin during their embryonic period and further research is warranted.

Optimization study of 2-hydroxyquinoxaline (2-HQ) biodegradation by Ochrobactrum sp. HQ1.

A novel aerobic gram-negative bacterial strain capable of utilizing 2-hydroxyquinoxaline (2-HQ) as sole source of carbon and energy was isolated from Indian agricultural soil and named as HQ1. Strain HQ1 was identified as Ochrobactrum sp. on the basis of morphology, physico-biochemical characteristics and 16S rRNA sequence analysis. The generation time of Ochrobactrum sp. HQ1 on 2-HQ at log phase is 0.71 h or 42.6 min. The degradation of 2-HQ by HQ1 under various physico-chemical parameters was analysed by HPLC and observed to be optimum with a high inoculum density (1.0 OD) at pH 7-8, temperatures 37-40°C and a high concentration of 2-HQ (500 ppm). Degradation of 2-HQ was also improved when additional nitrogen sources were used and this was attributed to the enhanced growth of the bacterium on the readily available nitrogen sources. Analysis of 2-HQ degradation by GC-MS resulted in elucidation of the degradation pathway for HQ1, a novel observation for aerobic Gram-negative bacteria. These findings are a possible indication of the application of HQ1 in the bioremediation of pesticide/metabolite contamination.

Variability in outcome and resource use in intensive care units.

OBJECTIVE: To examine variability in outcome and resource use between ICUs. Secondary aims: to assess whether outcome and resource use are related to ICU structure and process, to explore factors associated with efficient resource use. DESIGN AND SETTING: Cohort study, based on the SAPS 3 database in 275 ICUs worldwide. PATIENTS: 16,560 adults. MEASUREMENTS AND RESULTS: Outcome was defined by standardized mortality rate (SMR). Standardized resource use (SRU) was calculated based on length of stay in the ICU, adjusted for severity of acute illness. Each unit was assigned to one of four groups: "most efficient" (SMR and SRU < median); "least efficient" (SMR, SRU > median); "overachieving" (low SMR, high SRU), "underachieving" (high SMR, low SRU). Univariate analysis and stepwise logistic regression were used to test for factors separating "most" from "least efficient" units. Overall median SMR was 1.00 (IQR 0.77-1.28) and SRU 1.07 (0.76-1.58). There were 91 "most efficient", 91 "least efficient", 47 "overachieving", and 46 "underachieving" ICUs. Number of physicians, of full-time specialists, and of nurses per bed, clinical rounds, availability of physicians, presence of emergency department, and geographical region were significant in univariate analysis. In multivariate analysis only interprofessional rounds, emergency department, and geographical region entered the model as significant. CONCLUSIONS: Despite considerable variability in outcome and resource use only few factors of ICU structure and process were associated with efficient use of ICU. This suggests that other confounding factors play an important role.

Response of Cyprinus carpio (Linn) to sublethal concentration of cypermethrin: alterations in protein metabolic profiles.

Freshwater fish, Cyprinus carpio were exposed to sublethal concentration (1.2 microg l(-1)) of cypermethrin for 6, 12, 24 and 48 h to analyze various parameters of protein metabolism in functionally different tissues. Total, structural and soluble proteins showed decrement; where as free amino acids and the activities of protease, aspartate aminotransferase and alanine aminotransferase significantly increased in cypermethrin exposed fish. Interestingly, ammonia content decreased but urea and glutamine increased at all periods of exposure. It was also observed that alterations steadily increased with the period of exposure and exhibited tissue specificity. Thus variation in the protein metabolism of the fish exposed to cypermethrin indicates its toxic effect on the cellular metabolism thereby leading to impaired protein synthetic machinery.

GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) suppresses luteal steroidogenesis in the pregnant rat. We further demonstrated that the peripheral-type benzodiazepine receptor (PBR) and the steroidogenic acute regulatory protein (StAR) play key roles in cholesterol transport leading to steroidogenesis. The purpose of this study was to understand the cellular and molecular mechanisms involved in the suppression of luteal steroidogenesis leading to a fall in serum progesterone levels in GnRH-Ag-treated rats during early pregnancy. Pregnant rats were treated individually starting on day 8 of pregnancy with 5 microgram/day GnRH-Ag using an osmotic minipump. Sham-operated control rats received no treatment. At 0, 4, 8 and 24 h after initiation of the treatment, rats were killed and corpora lutea (CL) were removed for PBR mRNA, protein and radioligand binding analyses, immunoblot 1-D gel analysis of StAR, P450 scc and 3beta-hydroxysteroid dehydrogenase as well as 2-D gel analysis of StAR. The treatment decreased the luteal PBR mRNA expression at all time periods starting at 4 h compared with that in corresponding sham controls. GnRH-Ag also reduced, in the CL, the PBR protein/ligand binding, the StAR protein and P450 scc protein and its activity as early as 8 h after the treatment and they remained low compared with those in corresponding sham controls. The data from 2-D gel studies suggest that the majority of the decrease in StAR protein appears to be in the phosphorylated forms of StAR. Thus, we have demonstrated, for the first time, the presence of PBR and StAR in the pregnant rat CL and that the coordinated suppression of these proteins involved in the mitochondrial cholesterol transport along with P450 scc by GnRH-Ag leads to reduced ovarian steroidogenesis.

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.

In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis.

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.

Action of cypermethrin on tissue transamination during nitrogen metabolism in Cyprinus carpio.

Freshwater fish, Cyprinus carpio, was exposed to sublethal concentration (3 microg liter-1) of cypermethrin for 5 and 10 days to examine the changes in the transamination process during the formation of nitrogenous end products in four functionally different tissues, namely, gill, liver, brain, and muscle. Increases in total and soluble protein contents were noticed in all the tissues of exposed fish with a decrease in free amino acids and protease activity. Activity levels of both the transaminases, aspartate aminotransferase and alanine aminotransferase, and glutamate dehydrogenase were elevated, indicating active transamination and oxidative deamination. Attenuation of ammonia was consistent in both treatment groups. However, urea level decreased at the 5-day exposure period but increased by Day 10, manifesting the conversion of toxic ammonia to urea. Glutamine content was consistently raised upon exposure to the toxicant. In support of this, increases in glutamine synthetase and suppression of glutaminase were noticed. It clearly indicates that ammonia is not stored in the tissues in spite of active oxidative deamination when the fish is in a polluted environment. All the observations made demonstrate that the fish has adopted more than one compensatory mechanism during the process of transamination of nitrogenous products.

Role of phosphatases during transport and energy metabolism in Labeo rohita after exposure to cypermethrin.

Freshwater fish, Labeo rohita, were exposed to sublethal concentration (0.5 micrograms.L-1) of cypermethrin for 7 and 15 days to examine the bioenergetics in functionally four different tissues, namely, gill, liver, brain and muscle. Whole animal oxygen consumption was measured first and it was found to decrease in both the exposure periods (EPs), manifesting respiratory distress of the animal in toxic environment. Ionic regulation and energy requirements were also found to be altered under stress, as observed by the inhibition of both Na+/K+ and Mg2+ ATPases at 7d EP and elevation at 15d EP. Increase in glucose-6-phosphate dehydrogenase (G-6-PDH) was consistent with the increase in exposure time. Attenuation of acid and alkaline phosphatases were noticed in treated fish after 7 days but were close to normalcy at 15d EP. These results clearly indicate that the fish were affected at 7d EP but adapted to the toxic environment within 15 days. It shows that at this concentration cypermethrin is only moderately toxic and the animal has alternate pathways to derive energy and survive.

Cypermethrin-induced in vivo alterations in the carbohydrate metabolism of freshwater fish, Labeo rohita.

Aspects of carbohydrate metabolism under in vivo conditions were analyzed in functionally different tissues of the freshwater fish, Labeo rohita, exposed to a lethal (LC50/96 hr = 5.24 micrograms liter-1) and sublethal concentration (0.52 micrograms liter-1) of cypermethrin for 4 days. All exposed fish exhibited a hyperglycemic condition. An increase in tissue lactate with a decrease in pyruvate, total carbohydrates, and glycogen contents was noted. Activity of lactate dehydrogenase was elevated, indicating a shift toward anaerobiosis. TCA cycle enzymes, namely succinate dehydrogenase and malate dehydrogenase, were inhibited. In most cases changes were more pronounced during a lethal exposure compared to sublethal exposure period. The data indicate that the fish has adopted a compensatory mechanism to derive energy during pyrethroid toxicosis.

Protein metabolism in brain and muscle tissues of Mus booduga following repeated oral benzenehexachloride feeding.

Protein metabolism was studied in the brain and muscle tissues of mice, Mus booduga after administering orally 50 mg/kg body weight of benzenehexachloride (BHC) daily for 1, 5 and 15 days. Both tissues exhibited considerable decline in all the protein fractions such as total, soluble and structural proteins. This corroborates with the increased levels of free amino acids (FAA) and protease. To fortify these alterations, elevation in the activities of aspartate aminotransferase (AAT), alanine aminotransferase (AlAT) and glutamate dehydrogenase (GDH) were noticed. The two nitrogenous end products namely, ammonia and urea levels were also increased. These results clearly demonstrate the impairment of protein metabolism due to sublethal BHC toxicity.

Perturbations in nitrogen metabolic profiles in the tissues of fish, Labeo rohita exposed to fenvalerate.

Changes in the levels of nitrogen metabolic profiles in gill, brain, liver and muscle tissues of fish, Labeo rohita exposed to sublethal concentration (10 micrograms/L) of fenvalerate for 6, 12, 24 and 48 hours were studied. Ammonia content was decreased, whereas urea and glutamine levels were increased in the tissues of fenvalerate exposed fish. Changes were more pronounced with an increase in the period of exposure. Within the tissues, the alterations in excretory products were in the following order: liver greater than muscle greater than brain greater than gill. The survivability of fish in the polluted habitats might be due to the operation of compensatory mechanism in the metabolic profiles such as detoxification and transamination of more toxic ammonia by conversion to less toxic compounds like urea and glutamine.

Changes in the carbohydrate metabolism in the selected tissues of Mus booduga gray after BHC treatment.

Adult mice, Mus booduga were fed orally with bennzenehexachloride (BHC) at a dose of 50 mg/kg body weight every day for 1, 5 and 15 days. Significant decrease in the pyruvate content was observed at all periods of treatment. In support of this increase in lactate content and lactate dehydrogenase (LDH) activity was noticed in all the three tissues. Enzymes of TCA cycle namely isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) were inhibited suggesting abnormality in mitochondrial oxidative metabolism as a consequence of BHC toxicity.

Fenvalerate induced biochemical changes in the selected tissues of freshwater fish, Cyprinus carpio.

Alterations in the levels of some biochemical and enzymological parameters in brain, liver and muscle tissues of Cyprinus carpio exposed to sublethal concentration (10 micrograms/liter) of fenvalerate for 6, 12, 24 and 48 hr were studied. Total, structural and soluble proteins were decreased, whereas the free amino acids and the activities of protease, aspartate aminotransferase and alanine aminotransferase significantly increased in fenvalerate exposed fish. It is also observed that the changes were more pronounced with an increase in the period of exposure. Within the tissues, the alterations in biochemical and enzymological parameters was in the following order: liver greater than brain greater than muscle. The possible roles of fenvalerate induced changes in the fish are discussed.

Inhibition of Mg2+ and Na(+)-K+ ATPases in selected tissues of fish, Cyprinus carpio under fenvalerate toxicity.

The changes in the magnesium adenosine triphosphatase (Mg2+ ATPase) and sodium-potassium adenosine triphosphatase (Na(+)-K+ ATPase) in gill, brain, liver and muscle tissues of freshwater fish, Cyprinus carpio at 6, 12, 24 and 48 hr exposure periods were studied after subjecting to sublethal concentration (10 micrograms/lit) of fenvalerate. Mg2+ ATPase and Na(+)-K+ ATPase activities were inhibited in all the tissues of fenvalerate exposed fish. The per cent inhibition increased with increase in the period of exposure and the possible reasons for the inhibition patterns are discussed.